Human GC tissues and paired adjacent normal tissues were collected from 36 patients with GC who underwent radical gastrectomy at the Department of General Surgery, Shandong Provincial Hospital Affiliated Cheeloo College of Medicine of Shandong University, China. After surgical excision, the sample was quickly frozen in liquid nitrogen for experiments. This study was approved by the ethics committee of Shandong Provincial Hospital. Written informed consent is signed prior to specimen collection. The collection of gastric cancer and paracancer tissue and its use for research purposes was approved by the Institutional Review Board of the Shandong Provincial Hospital affiliated to Shandong University, Shandong, China.
Six gastric cancer cell lines (BGC-823, HGC-27, MGC-803, SGC-7901, MKN-45, AGS) and GES-1 cell lines were purchased from Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (10% FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, Grand Island, NY,USA) was incubated in moist air at 37°C and 5% CO2.
Hsa-miR-875-5p mimics and mimics negative control, hsa-miR-875-5p inhibitor and inhibitor negative control were purchased from Gemma. (Shanghai, China). At least 24 hours before transfection, the cells were cultured in complete medium without antibiotics. Cells were planted in six-well plates, when the cell fusion degree reached 50%-70%, the cells were washed with 1x PBS (PH7.4), 50nM miR-875-5p mimics or miR-mimic NC and 100nM miR-875-5p inhibitors or miR-inhibitor NC were transfected into AGS and MKN-45 cells through Lipofectamine™2000 (Invitrogen). Si-USF2 and si-NC were purchased from Genome (Shanghai, China) and transfected as above.
RNA extraction and RT-qPCR
Total RNA was extracted from GC tissues and cells using Trizol reagent (Takara, Japan) according to the manufacturer's instructions. The cDNA was synthesized using the Evo M-MLV RT Premix for qPCR reagent (AG, China). QRT-PCR was performed on Roche LightCycler 480 II fluorescent quantitative PCR instrument with qPCR SYBR Green Pro Taq HS Master Mix(AG). β-actin was used as the endogenous control. The primers used in this study are as follows: USF2 forward: 5'-AAAGGAGGGATCCTGTCCAA-3', USF2 reverse: 5'-CAGGGCGTTCTCATTCTTCA-3'; β-actin forward: 5'-GCATCGTCACTGGGGAC-3' and β-actin reverse: 5'-ACCTGG CCGTCAGGCAGCTC-3'. In addition, unchained curves were used to evaluate nonspecific amplification. QRT-PCR reaction procedures are as follows: 95℃ for 30s, 40 cycles at 95°C 5 s and 60°C 30 s. All procedures were carried out in triplicate and relative expression was calculated by the 2−ΔΔCT method.
Total RNA was extracted as mentioned above. According to the manufacturer's instructions, using the Mir-X miRNA first-Strand Synthesis Kit (Takara, Japan), RNA (2 µg) was converted to cDNA. PCR reaction was performed using TB Green® Premix Ex TaqTM II (Takara, Japan), and U6 was used as the endogenous control. The primers of Mir-875-5p and U6 were purchased from RiboBio (RiboBio Co., Ltd, Guangzhou, China), the primer sequences used in this study were as follows; has-miR-875-5p forward:5’-GCGGGCGGTATACCTCAGTTTTAT-3’, reverse 5’-ATCCAGTGCAGGGTCCGAGG-3’; U6 forward: 5′-CTCGCTTCGGCAGCACA-3′; U6 reverse: 5′-AACGCTTCACGAATTTGCGT-3′. 2-ΔΔCt method was used to calculate the relative expression level. All procedures were also performed in triplicate.
Protein extraction and Western blot
72h after transfection, the cells were flushed with cold PBS, then RIPA buffer（Beyotime, Shanghai，China）containing PMSF (SolarBio, Beijing, China) and phosphatase inhibitors (SolarBio, Beijing, China) was used for cracking. The protein concentration was calculated using the BCA Protein Assay Kit (Beyotime, Shanghai，China) and the protein was separated by SDS-PAGE using a 10% and 12% polyacrylamide gel (20μg per sample). Proteins are transferred to the immobilon-NC membrane by electrotransfer. The imblotted membrane was sealed in 5% skimmed milk diluted with TBST, and then incubated with appropriate primary antibodies (anti-USF2, anti-p21, anti-p57, anti-E-cadherin, anti-Vimentin, anti-smad2, anti-phospho-smad2, anti-smad3, anti-phospho-smad3 and anti-GADPH obtained from CST) at 4°C for 12 h.
Dual-luciferase reporting assay
Dual luciferase assay was used to further verify the targeting relationship of miR-875-5p and USF2. Bioinformatics analysis predicted that the possible binding site of miR-875-5p was the 3 'UTR site 595-601 of USF2 mRNA. AGS was grown in 1640 medium containing 10% FBS and transfected with psicheck2-Husf2-3 'UTR reporter plasmid and human miR875-5p mimics using Lipofectamine™2000. The dual luciferase reporter gene system was used to detect whether human microRNAs bind and regulate USF2.
Cell growth was measured using cell proliferation reagent CCK-8 (MCE). Cells were inoculated into a 96-well plate (Corning Costar, Corning, NY) at a concentration of 2.0×103 per well, as per manufacturer's instructions, and 10μLCCK8 was added to each well at harvest. One hour after CCK8 was added, cell viability was determined by measuring the absorbance of the transformed dye at 450nM.
The transfected cells were seeded into a 96-well plate at a concentration of 1.0×104/well. The EDU solution (Reagent A) was diluted with cell complete medium in a ratio of 1000:1 to prepare an appropriate amount of 50μM EDU medium according to the manufacturer's instructions. Add 100μL 50 μm EDU medium to each well and incubate for 2 hours, then discard the medium; PBS washed the cells twice, 5 minutes each time. Add 50μl cell fixation solution (PBS containing 4% paraformaldehyde) to each well and incubate at room temperature for 30 min, then discard the fixation solution; Add 50μl 2 mg/mL glycine to each well, and incubate on decolorizing shaker for 5 min, then discard the glycine solution; Add 100μl PBS to each well, wash with decolorizing shaker for 5min, discard PBS; Add 100μl penetrant (0.5% TritonX-100 PBS) to each well and incubate in decolorizing shaker for 10 min. PBS cleaning once, 5 minutes; Add 1X Apollo® dyeing reaction solution of 100μl to each well, and incubate for 30 min in dark, at room temperature, decolorizing shaker, then discard the dyeing reaction solution. Add 100μl penetrant (0.5% TritonX-100 PBS) to decolorizing shaker, wash it 3 times, 10 min each time, discard penetrant; Add 100μl methanol to each well and clean it twice, 5min each time; PBS cleaning once, 5 minutes each time; Add 100μl 1X Hoechst 33342 reaction solution to each well, and incubate in dark, at room temperature and decolorized shaker for 30 min. Then discard the dyeing reaction solution. Add 100μl PBS to each well and clean 3 times; Add 100μl PBS to each well, save. The results were observed using a high-content imaging system.
Transwell migration/invasion assay
MKN-45 and AGS cells grew to approximately 70% confluence in RPMI 1640 containing 10% fetal bovine serum and then transfected. After 24 hours, the cells were digested by trypsin and then washed once with PBS. To measure cell migration, culture inserts with an 8 mm aperture (Transwell; CoStar, High Wycombe, UK) were placed in the holes of the 24-well plate and the upper and lower chambers were separated. In the lower chamber, add RPMI 1640 containing 20% FBS, 600μl. Then, serum-free medium containing 5×104 cells was added to the upper compartment for migration assay, and 1×105 cells were used for matrix gel invasion assay. After incubation at 37°C and 5% CO2 for 24 h, the cells in the upper compartment were removed with a cotton swab. The cells that invaded the base membrane of the inserts were fixed in 100% methanol for 10 min, stained with 1% crystal violet for 20 min, rinsed in PBS, and examined microscopically. Each experiment was performed at least three times.
Wound healing assay
To evaluate the motility of GC cells. A total of 1×106 cells/Wells were inoculated in a 6-well plate and cultured overnight, then transfected with miR-875-5p mimics or NC, miR-875-5p inhibitor or NC and si-USF2 or NC. Eight hours later, the confluent cell monolayers were scraped with a sterile pipette head and the plates were washed twice with PBS and cultured for 24 hours before adding fresh serum-free medium. An image of the plate is taken under a microscope. The clearance size was analyzed with Image J software.
Tumorigenesis in nude mice
BALB/c nude mice (male, 4-6 weeks old, 16-20 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). All animal experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of Provincial Hospital Affiliated to Shandong University. MiR-875-5p NC and miR-875-5p mimics were transfected into MKN-45 cells, and 5×105 MKN-45 cells in logarithmic growth phase were suspended in 100μL phosphate buffer, and then seeded subcutaneously into the right axillary of nude mice. The experiments were divided into two groups on average (miR-875-5p NC group and miR-875-5p mimics group, n =6), tumor size was monitored by measuring length (L) and width (W) with a vernier caliper every 4 days, and volume was calculated using the following formula: (L×W2) / 2. The mice were fed for 28 days. Tumors were sacrificed and collected. Tumor volume and weight was measured for analysis. Animal experiments conformed to the standards set by the Declaration of Helsinki, and were approved by the ethics review Committee of Shandong Provincial Hospital affiliated to Shandong University, Shandong, China.
Immunohistochemical staining of xenograft tumors tissue
Tumor sections were incubated overnight with commercial rabbit polyclonal antibodies against USF at 1:50 dilution at 4°C. Then, the slices were diluted with horseradish peroxidase (HRP) antibody (1:100; Invitrogen, Thermo Fisher, US) was conjugated at room temperature for 2 h and then covered with DAB (SP kit (rabbit streptavidin-biotin method detection system), ZSGB-BIO, China). After rinsing the colored plates with water for a period of time, they were soaked in hematoxylin and dyed, then dehydrated and sealed. Subsequently, all fields of view were observed under a light microscope (Tissue FAXS Systems, Austria).
The results are presented as means ± SD. Statistical significance was measured by multiple comparisons using Student’s t-test with a significance level of p < 0.05.